Enhanced stability and clinical absorption of a form of encapsulated vitamin A for food fortification

Significance Fortifying common daily foods is an ideal solution to decrease the prevalence of vitamin A (VitA) deficiency. However, VitA food fortification is challenging due to its poor stability under heat,light, moisture, and oxidative conditions and its poor miscibility with water or centrally processed foods, such as flour. It is crucial to address both stability and bioavailability to ensure the efficacy of VitA food fortification. Here, we developed a VitA encapsulation technology that is produced by a commercial process, provides effective protection during storage and cooking, and is bioavailable in humans.

The oral administration was delivered by gavage to the restrained rats using a ball-tipped stainless-steel gavage needle. The residual T-VitA in the syringe and gavage needle was quantified by scintillation counter to calculate the actual feeding amount of T-VitA for each rat. After 24 hours, the rats were euthanized by CO2, and their stomach, small intestine, liver, lung, spleen, kidney, heart and eye tissues were collected for analysis.
T-VitA radioactivity measurement. The radioactivity in the samples was quantified by liquid scintillation counting with a TriCarb liquid scintillation analyzer (with a counting efficiency of 36% for 3 H in Hionic-Fluor liquid scintillation cocktail and 65% for 3 H in Ultima Gold TM F liquid scintillation cocktail). To determine the loading of T-VitA in MPs, the T-VitA-BMC was first dissolved in 1 mL dichloromethane, and then 5 μL of the solution was mixed with 10 mL Ultima Gold TM F liquid scintillation cocktail (PerkinElmer Inc.). Standard solutions were prepared by mixing the 1 mCi/mL T-VitA solution in ethanol with the same amount of liquid scintillation cocktail. A blank was prepared by mixing dichloromethane with the Ultima Gold TM F liquid scintillation cocktail. The solution was allowed to set for at least 1 hour before reading. The concentration of radioactivity in the sample solution, , was calculated by the equation where 0 is the concentration of standard solution in μCi/mL, 0 is the counts per minute for the standard solution, is the counts per minute for the blank, and is the counts per minute for the sample solution. For quantification of T-VitA, the blood and tissue samples were dissolved with SOLVABLE TM (PerkinElmer Inc.) following a standard protocol. 1 mL T-VitA solution in SOLVABLE TM was mixed with 10 mL Hionic-Fluor liquid scintillation cocktail as sample solution. 200 μL blood from a rat that was not fed T-VitA was prepared in the same manner and served as the blank solution. A standard solution was prepared from a known amount of T-VitA in 200 μL non-radioactive blood. The concentration was calculated using Equation 1. The total blood volume for each rat was calculated based on the total weight of each rat. The percentage of T-VitA in blood to the total feeding is calculated by equation where is the concentration of radioactivity in the blood in μCi/mL, is the total blood volume of the rat in mL, and is the actual amount of radioactivity fed to the rat. Similarly, whole tissues were weighed, and the percentage of T-VitA in the tissues to the total feeding is calculated. The absorbed T-VitA is the sum of the T-VitA in liver, small intestine, lung, kidney, heart, spleen and eye.

Methods for human clinical trial
Subject inclusion criteria. Subject inclusion criteria were as follows: 18-30 years of age, healthy, premenopausal females, a BMI between 18.5-29.9 kg/m 2 , no dietary supplement intake, and maintains a regular diet and physical activity pattern while abstaining from restricted foods and vigorous exercise. Exclusion criteria specifies things in opposition to the inclusion criteria as well as recent history of anemia, recent blood transfusions or donations, a plasma homocysteine concentration >12 µmol/L, or a presence/history of cardiac, renal, hepatic, endocrine, cardiovascular, pulmonary, pancreatic, neurologic, immunologic, dermatologic, rheumatic, gastrointestinal, active infection, cancerous, and/or biliary conditions. Further exclusions include VitA deficiency, folate deficiency, recent trauma/surgical events, uncontrolled hypertension, chronic use of anti-inflammatory drugs/antacids/medicines that interfere with VitA/FA/Fe absorption, use of hormone therapy, pregnancy, an allergy/sensitivity to any of the food ingredients used in the study meals, or alcohol abuse.
Randomization and masking in clinic study. A randomization sequence prepared by a statistician was uploaded onto Medrio eCRF platform (Medrio Inc, San Francisco, CA). When a subject was determined to be eligible for the study, a randomization number was assigned to the subject through randomization module of the Medrio platform. The randomization number was recorded in the subject's source documentation.
Sample size in clinic study. The evaluable sample sizes calculated were N = 30 for folic acid and N = 23 for vitamin A. To account for attrition, a total of 37 subjects were randomized.
Sample size calculation in clinic study. For vitamin A metabolites, sample size calculation was performed in consultation with Dr. Georg Lietz (Newcastle University, UK) using the following parameters: i) Reference group: Mean AUC = 0.21, SD = 0.09; ii) Estimated values for comparator group: Mean AUC = 0.147, SD = 0.09, assuming 30% difference with reference group and SD kept constant; iii) Alpha = 0.05 and iv) Power = 90%. The resulting evaluable sample size is 23 subjects. For folate, sample size calculation was done assuming similar mean and SD AUC from previous study (34) with the following parameters: i) Reference group: Mean AUC = 0.21, SD = 0.09; ii) Estimated values for comparator group: Mean AUC = 146 nmol/Lh, SD = 58 nmol/Lh, assuming 20% difference with reference group and difference between groups is 10 nmol/Lh; iii) Alpha = 0.05 and iv) Power = 80%. The resulting evaluable sample size is 30 subjects.
Procedures in clinic study. Each subject consumed one meal within 10 min per visit, which consisted of a fortified bread bun served with 10 g (± 2.5%) of a soybean oil spread and 240 mL of water. As shown in Figure S5, each subject completed a total of 5 visits to consume one of each of the 5 test meals in a randomized, blinded order. The meals, the fortificants used in the meals, and the dose levels are listed in Table 1. In all meal groups, 1.28 mg (the recommended daily allowance for VitA for pregnant women) of isotopically-labeled VitA (vitamin A palmitate (12,13,14, C4) stabilized by 0.5% butylated hydroxytoluene (BHT); produced at Buchem B.V., The Netherlands) was used to fortify the meal. The free VitA in Group 5 was added to the oil spread and therefore was not baked; it is the uncooked control. Group 1 is the unencapsulated free VitA that was baked into the bread with free FA; it is the cooked control with stabilizer BHT. The VitA in Group 2 to 4 is encapsulated in VitA-BMC-S MPs with or without codelivery of encapsulated Fe (formulation in Table S4) and unencapsulated free folic acid. Following a typical Senegalese bread recipe, each bun consisted of 100 g of bread dough (wheat flour, salt, dry yeast, water, and fortificants) that was allowed to rise for 45 min at 30 o C and 80% relative humidity. The buns were then baked at 190 o C for 20 min. The fortificants were specifically kneaded into the center portion of the bread dough buns to reduce VitA loss.
A 48-h Diet Record was dispensed to confirm compliance with adherence to a low-folate/folic acid diet throughout the 48-h period prior to test visits. A half hour prior to the study meal consumption, the subject gave blood for baseline measurements of retinyl palmitate and plasma folate. After meal consumption, the subject gave blood at designated timepoints in order to fulfill the needs for plasma folate analyses at t = 1, 1.5, 2, 3, 4, 6, and 8 h ± 5 min and for plasma retinyl palmitate analyses at t = 2, 4, 6, 8, 10, and 12 h ± 5 min. No food was ingested after study meal consumption for the subsequent 4 h period, but ad libitum water consumption was allowed for the remainder of the visit following the 2 h blood draw. Subjects were administered a standard low-folate/folic acid, low-vitamin A lunch (immediately following the t = 4 h blood draw), a standard low-folate/folic acid, low-vitamin A snack (immediately following the t = 6 h blood draw) and a standard low-folate/folic acid, low-vitamin A dinner (at t = 10 h). Subjects were instructed to consume the meal/snacks within 30 min and to eat until comfortably full. The meals and snack (choices and amounts) were replicated at subsequent visits. A washout window of 5-7 days followed each test meal visit, and each subject was designated to complete all 5 meal groups.
Outcomes in clinic study. The retinyl palmitate was extracted from the plasma samples using hexanes and quantified by LC-MS-MS. The folate levels in the plasma samples were determined using the commercially available microbiological kit (ALPCO, ref# KIF005) in a 96-well format. Samples were analyzed by Eurofins Craft Technologies. Outcome variables included the following: i) positive incremental area under the curve (AUC) for plasma folate from 0 to 8 h, ii) AUC for plasma 13 C4-retinyl palmitate from 0 to 24 h, iii) maximal concentration (Cmax) of plasma 13 C4-retinyl palmitate and plasma folate, and iv) time to maximal concentration (tmax) of plasma retinyl palmitate and folate. For plasma folate, the bioequivalence and difference tests were performed by comparing: Group 1 vs. Group 5, Group 2 vs. Group 5, Group 2 vs. Group 4, Group 1 vs. Group 4, and Group 1 vs. Group 2. For plasma retinyl palmitate, the bioequivalence and difference tests were performed by comparing: Group 1 vs. Group 5, Group 2 vs. Group 5, Group 3 vs. Group 5, Group 4 vs. Group 5, Group 2 vs. Group 4, and Group 1 vs. Group 2. For all the aforementioned comparisons, only difference test was performed for tmax.
Statistical analysis in clinic study. All statistical analyses were conducted using SAS for Windows (version 9.2, or higher, Cary, NC) and/or R 3.3.1 (R Core Team 2016). Thirty-one subjects completed all 5 test meals of the study (in addition to the 31, 1 subject was lost to an adverse event that was medically deemed unrelated to the study, 1 subject was lost to withdrawal of consent, and 4 subjects were lost because they failed to follow-up). Intent-to-treat population data with outliers was used for all analyses (n=31). The difference between a pair of meal groups was assessed using Wilcoxon signed-rank test when the normality assumption was not satisfied. When the normality assumption was satisfied, difference was assessed using unpaired t-test (for equal variance) and Welch's t-test (for unequal variance).
Role of funding source in clinic study. The funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Adverse events and other clinical observations in clinic study.
Adverse event (AE) inquiries occurred at the beginning (Visits 2 through 11; days 0 through 29) and end (Visits 2, 4, 6, 8, and 10; days 0, 7, 14, 21, and 28) of the study visits. Subjects were asked an open-ended question (e.g. "since you were last asked, have you noticed any changes in the way you feel?") for each AE assessment.
A total of 28 clinical observations were reported by 14 subjects during the study. Six of these (nausea, vomiting, bloating, diarrhea, flatulence) were judged as possibly related to the study product and the others were judged as not related to study product by the Clinical Investigator. All adverse events were judged to be mild to moderate, except for two (nausea and vomiting) reported by Subject 002 which were judged to be serious by the Clinical Investigator. Subject 002 experienced nausea and vomiting during Visit 2 and did not complete the visit. During a follow up call, the subject reported being admitted to the hospital and provided IV fluids and treated for nausea/vomiting. Additional subsequent attempts to obtain follow-up information failed due to subject being non-responsive and subject was considered lost to follow-up.      S5. A randomized controlled crossover study was designed to assess the VitA bioavailability of a fortified bread meal. The study includes one screening visit (Visit 1; day -7) and five test periods [Test Period 1 (Visits 2 and 3; days 0 and 1); Test Period 2 (Visits 4 and 5; days 7 and 8); Test Period 3 (Visits 6 and 7; days 14 and 15); Test Period 4 (Visits 8 and 9; days 21 and 22); and Test Period 5 (Visits 10 and 11; days 28 and 29)].